Sulfite Oxidase (SO) is a molybdenum cofactor dependent enzyme that catalyzes the oxidation of sulfite to sulfate as the final step in the degradation of the sulfur-containing amino acids, cysteine and methionine. SO is physiologically vital in human metabolism, and hereditary sulfite oxidase deficiency results in severe neurological damage with symptoms that include dislocation of the ocular lenses, attenuated growth of the brain, mental retardation and early death. SO deficiency results either from a defect in the synthesis of the molybdenum cofactor or from various point mutations with the enzyme. While SO's from several sources have been previously studied, there is no comprehensive set of structural, spectroscopic and reactivity data from a common source. The primary goal of the research presented in this proposal is to gain greater insight into the function and mechanism of SO and the effect of point mutations through an integrated series of biochemical and biophysical studies of SO from Mus musculus (house mouse), which is readily expressed in DL41 plus E. coli cells. Mouse SO is 82% identical with the human enzyme, but has not been previously investigated extensively. The primary tools for investigating the properties of mutants (variants) of mouse SO will be: 1) Laser flash photolysis studies of intramolecular electron transfer (IET) between the molybdenum domain and the b type heme domain; 2) high resolution variable frequency pulsed EPR experiments to determine the structure of the transient catalytic molybdenum center through nuclear couplings (1H, 2H, 31P, 17O and 33S); 3) crystallization and X-ray structure determination as a function of pH and anions in the media. Despite numerous attempts in other laboratories, intact human SO has proven difficult to crystallize. To date the only crystal structure for intact animal SO is the 1997 structure of wild-type chicken SO. For human SO, the only available structural information is for the isolated heme domain. Plant SO, from Arabidopsis thaliana only has a molybdenum domain. Thus, there is a need for an animal system that has a high sequence homology to human SO and for which substantial quantities of enzyme variants can be systematically produced for study. Mouse SO meets these criteria and preliminary data on expression of the protein show the feasibility of the proposed studies. [unreadable] [unreadable] [unreadable]